Picornaviruses as well as other positive strand RNA viruses utilize sequences in the 5' noncoding regions (5' NCRs) of their genomic RNAs to bind host cell and viral proteins to carry out important functions during their intracellular replication cycles. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA secondary structure in their 5' NCRs required for internal ribosome entry during translation initiation. These structures (i.e., IRES elements) bind several host RNA binding proteins, including poly(rC) binding protein 2 (PCBP2). Binding of PCBP2 is required for poliovirus translation initiation, a process that also requires the function of a nucleo-cytoplasmic shuttling protein, SRp20. The first aim of this proposal is to identify components of translation initiation complexes assembled with PCBP2-bound poliovirus IRES sequences. Genetic, biochemical, and confocal microscopy experiments will test the hypothesis that SRp20 acts as a molecular bridge between PCBP2 bound to the poliovirus IRES and specific elements of the cellular translation initiation apparatus. In addition to its role in poliovirus translation, cellular protein PCBP2 is required for negative-strand viral RNA synthesis. During poliovirus infection of HeLa cells, PCBP2 is cleaved by a viral proteinase to generate a truncated protein that is unable to function in translation but retains its role in RNA replication. The second aim of the proposal will utilize cell culture and in vitro translation/RNA replication approaches to determine if this cleavage is responsible for clearing poliovirus genomic RNAs of translating ribosomes prior to the onset of viral RNA synthesis, providing a mechanistic switch for these two competing functions utilizing positive- strand RNA templates. Results from these proposed studies will provide new mechanistic insights into the interplay between translation functions and picornavirus RNA replication. In a new direction for this project, IRES elements have been identified in the long 5' NCRs of two cellular mRNA molecules, one that encodes a voltage-gated potassium channel (Kv1.4) and one that encodes a transcription factor (LEF-1). The experiments proposed in the third aim will examine the nature of RNP complexes formed with the 5' NCRs of these two cellular mRNAs to define the determinants of IRES functions for translation of mRNAs that, unlike picornavirus RNAs, are synthesized in the nucleus of cells and must be transported to the cytoplasm prior to their association with 40S ribosomal subunits. Results from these latter studies will provide important side-by-side comparisons with picornavirus IRES functions and new insights into how eukaryotic cells initiate translation from mRNAs with long, highly-structured 5' NCRs.